Alzheimer""s disease is a progressive and ultimately fatal form of dementia that affects a substantial portion of the elderly population. Definitive diagnosis at autopsy relies on the presence of neuropathological brain lesions marked by a high density of senile plaques. These extracellular deposits are found in the neo-cortex, hippocampus and amygdala as well as in the walls of the meningeal and cerebral blood vessels. The principal component of these plaques is a 39 to 43 residue xcex2-amyloid peptide. Each plaque contains approximately 20 fmole (80 picograms) of this 4 kDa peptide (Selkoe et al., J. of Neurochemistry 46: 1820 (1986)). Apolipoprotein E and neurofibrillary tangles formed by the microtubule-associated tau protein are also often associated with Alzheimer""s disease.
xcex2-amyloid is proteolytically cleaved from an integral membrane protein called the xcex2-amyloid precursor protein. The gene which codes for this protein in humans is found on chromosome 21 (St George-Hyslop et al., Science 235: 885 (1987), Kang et al., Nature 325: 733 (1987)). Numerous cultured cells and tissues (eg. brain, heart, spleen, kidney and muscle) express this xcex2-amyloid precursor protein and also secrete the 4 kDa xcex2-amyloid fragment into culture media, apparently as part of a normal processing pathway.
While it is difficult to establish an absolute causal relationship between xcex2-amyloid or the plaques it forms and Alzheimer""s disease, there is ample evidence to support the pathogenic role of xcex2-amyloid. For example, patients with Down""s syndrome have an extra copy of the xcex2-amyloid precursor protein gene due to trisomy of chromosome 21 (St George-Hyslop et al., Science 235: 885 (1987), Kang et al., Nature 325: 733 (1987)). They correspondingly develop an early-onset Alzheimer""s disease neuropathology at 30-40 years of age. Moreover, early-onset familial Alzheimer""s disease can result from mutations in the xcex2-amyloid precursor protein gene which fall within or adjacent to the xcex2-amyloid sequence (Hardy, J., Nature Genetics 1: 233 (1992)). These observations are consistent with the notion that deposition of xcex2-amyloid as plaques in the brain are accelerated by an elevation in its extracellular concentration (Scheuner et al., Nature Med. 2: 864 (1996)) The finding that xcex2-amyloid is directly neurotoxic both in vitro and in vivo (Kowall et al., Proc. Natl. Acad. Sci. 88: 7247 (1991)), suggest that soluble aggregated xcex2-amyloid, not the plaques per se, may produce the pathology.
Observations have indicated that amyloid plaque formation may proceed by a crystallization type mechanism (Jarrett et al., Cell 73: 1055 (1993)). According to this model, the seed that initiates plaque nucleation is an xcex2-amyloid which is 42 or 43 amino acids long (Axcex21-43). The rate-determining nucleus formed by Axcex21-43 or Axcex21-42 allows peptides Axcex21-40 or shorter to contribute to the rapid growth of an amyloid deposit. This nucleation phenomenon was demonstrated in vitro by the ability of Axcex21-42 to cause the instantaneous aggregation of a kinetically stable, supersaturated solution of Axcex21-40. That finding has led to the possibility that Axcex21-40 might be relatively harmless in the absence of the nucleation peptides Axcex21-42 or Axcex21-43. Indeed, elevated levels of these long peptides have been found in the blood of patients with familial Alzheimer""s disease (Scheuner et al., Nature Med. 2: 864 (1996)). Moreover, Axcex21-42 or Axcex21-43 was found to be the predominant form deposited in the brain plaques of many Alzheimer""s disease patients (Gravina et al., J. of Biol. Chem. 270: 7013 (1995)).
Given the central role played by xcex2-amyloid, it has become increasingly important to understand the interrelationship between the different pools of these molecules in the body. Free xcex2-amyloid present in the blood most likely arises from peptide released by proteolytic cleavage of xcex2-amyloid precursor protein present on cells in the peripheral tissues. Likewise most of the free xcex2-amyloid found in the brain and cerebrospinal fluid is probably derived from peptide released by secretase cleavage of xcex2-amyloid precursor protein expressed on brain cells. The peptides are identical regardless of origin, and the results from several studies suggest an intercommunication between these pools.
One aspect of the present invention is an antibody which catalyzes hydrolysis of xcex2-amyloid at a predetermined amide linkage. In one embodiment, the antibody preferentially binds a transition state analog which mimics the transition state adopted by xcex2-amyloid during hydrolysis at a predetermined amide linkage and also binds to natural xcex2-amyloid with sufficient affinity to detect using an ELISA. In another embodiment, the antibody preferentially binds a transition state analog which mimics the transition state adopted by xcex2-amyloid during hydrolysis at a predetermined amide linkage, and does not bind natural xcex2-amyloid with sufficient affinity to detect using an ELISA. Antibodies generated are characterized by the amide linkage which they hydrolyze. Specific antibodies include those which catalyze the hydrolysis at the amyloid linkages between residues 39 and 40, 40 and 41, and 41 and 42, of xcex2-amyloid.
Another aspect of the present invention is a vectorized antibody which is characterized by the ability to cross the blood brain barrier and is also characterized by the ability to catalyze the hydrolysis of xcex2-amyloid at a predetermined amide linkage. In one embodiment, the vectorized antibody is a bispecific antibody. Preferably, the vectorized antibody has a first specificity for the transferrin receptor and a second specificity for a transition state adopted by xcex2-amyloid during hydrolysis. Specific vectorized antibodies include those which catalyze the hydrolysis at the amyloid linkages between residues 39 and 40, 40 and 41, and 41 and 42, of xcex2-amyloid.
Another aspect of the present invention is a method for sequestering free xcex2-amyloid in the bloodstream of an animal by intravenously administering antibodies specific for xcex2-amyloid to the animal in an amount sufficient to increase retention of xcex2-amyloid in the circulation. Therapeutic applications of this method include treating patients diagnosed with, or at risk for Alzheimer""s disease.
Another aspect of the present invention is a method for sequestering free xcex2-amyloid in the bloodstream of an animal by immunizing an animal with an antigen comprised of an epitope which is present on xcex2-amyloid endogenous to the animal under conditions appropriate for the generation of antibodies which bind endogenous xcex2-amyloid. Therapeutic applications of this method include treating patients diagnosed with, or at risk for Alzheimer""s disease.
Another aspect of the present invention is a method for reducing levels of xcex2-amyloid in the brain of an animal by intravenously administering antibodies specific for endogenous xcex2-amyloid to the animal in an amount sufficient to increase retention of xcex2-amyloid in the circulation of the animal. In one embodiment, the antibodies are catalytic antibodies which catalyze hydrolysis of xcex2-amyloid at a predetermined amide linkage. The antibodies may be either monoclonal or polyclonal. In one embodiment, the antibodies specifically recognize epitopes on the C-terminus of xcex2-amyloid1-43.
Another aspect of the present invention is a method for reducing levels of xcex2-amyloid in the brain of an animal, by immunizing the animal with an antigen comprised of an epitope which is present on endogenous xcex2-amyloid under conditions appropriate for the generation of antibodies which bind endogenous xcex2-amyloid. In one embodiment, the antigen is a transition state analog which mimics the transition state adopted by xcex2-amyloid during hydrolysis at a predetermined amide linkage. In a preferred embodiment, the antigen is comprised of Axcex210-25. Preferably, the antibodies generated have a higher affinity for the transition state analog than for natural xcex2-amyloid, and catalyze hydrolysis of endogenous xcex2-amyloid.
Similar methods which utilize or generate antibodies which catalyze the hydrolysis of xcex2-amyloid for reducing levels of circulating xcex2-amyloid in an animal, and also for preventing the formation of amyloid plaques in the brain of an animal, are also provided. Also, methods for disaggregating amyloid plaques present in the brain of an animal by utilizing or generating antibodies which catalyze the hydrolysis of xcex2-amyloid are provided.
Another aspect of the present invention is a method for disaggregating amyloid plaques present in the brain of an animal by intravenously administering vectorized bispecific antibodies to the animal in an amount sufficient to cause significant reduction in xcex2-amyloid levels in the brain of the animal. The vectorized bispecific antibodies are competent to transcytose across the blood brain barrier, and have the ability to catalyze hydrolysis of endogenous xcex2-amyloid at a predetermined amide linkage upon binding. Preferably, the vectorized bispecific antibodies specifically bind the transferrin receptor.
Another aspect of the present invention is a method for generating antibodies which catalyze hydrolysis of a protein or polypeptide by immunizing an animal with an antigen comprised of an epitope which has a statine analog which mimics the conformation of a predetermined hydrolysis transition state of the polypeptide, under conditions appropriate for the generation of antibodies to the hydrolysis transition state. This method can be used to generate catalytic antibodies to xcex2-amyloid. A similar method, which utilizes reduced peptide bond analogs to mimic the conformation of a hydrolysis transition state of a polypeptide is also provided.